The invention relates to the isolation of nucleic acid from cellular sources, and more particularly the isolation of high molecular weight genomic DNA from biological fluids and bacterial artificial chromosomes from transformed bacterial cultures.
Isolated nucleic acid, and in particular, isolated high molecular weight DNA, has a variety of uses in molecular biology, biotechnology and clinical research. For example, isolated DNA is useful in a number of molecular biology techniques, including polymerase chain reaction (PCR), DNA hybridization, restriction enzyme digestion, DNA sequencing, and array-based experiments. With regard to biotechnology, isolated DNA is useful in the development of genetically engineered recombinant proteins and in identifying potential new therapeutic targets. In the clinical setting, isolated DNA is useful in the identification of genetic disorders and in the diagnosis of bacterial and/or viral infections. As such, there is a need for simple and reliable methods for isolating DNA, and in particular, for isolating high quality, high molecular weight DNA.
The most commonly used method for isolating DNA from a DNA source, e.g., blood, saliva, bacterial cultures, etc., involves lysing the DNA source with a combination of a proteolytic enzyme and a detergent followed by extracting the mixture with an organic solvent, e.g., phenol and chloroform, so that the DNA enters the aqueous phase and the hydrolyzed products enter the organic phase. The DNA in the aqueous phase is then precipitated by the addition of alcohol. However, these organic extraction methods are laborious and time consuming and require the use of phenol (or other organic solvents), which are typically toxic and, therefore, a safety hazard.
In another approach, the DNA is isolated by lysing the DNA source with a chaotropic substance, for example guanidinium salt, urea and sodium iodide, in the presence of a DNA binding solid phase. The released DNA is bound to the solid phase in a one step reaction, where the beads are washed to remove any residual contaminants. Although these methods have proven to be less time consuming and toxic, they have resulted in a moderate level of DNA shearing and some level of contamination.
In a further approach, the DNA is isolated from a starting source by mixing the starting source with a cationic detergent, which forms a hydrophobic complex between the DNA and detergent. The hydrophobic complex is separated from the solubilized contaminants and the DNA recovered by addition of a salt. As above, this approach has proven to be much less time consuming, but does result in some level of DNA shearing and contamination. Against this backdrop the present invention has been developed.
The present invention provides novel methods and kits for the isolation of nucleic acid from a nucleic acid containing starting material. One method of the present invention includes mixing the starting material with a lysing and denaturing substance for release of the nucleic acid from the starting material; separating the mixture into a first portion and a second portion, where the first portion has a greater amount of the nucleic acid than the second portion; adding an alcohol and detergent substance to the first portion to precipitate the nucleic acid onto a nylon membrane; and re-suspending the isolated nucleic acid off of the nylon membrane in a re-suspension buffer.
These and various other features as well as advantages that characterize the invention will be apparent from a reading of the following detailed description and a review of the associated figures.